The invention concerns a method and a device for carrying out biochemical reactions especially for polypeptide biosynthesis and for the coupled in vitro transcription and translation of proteins in a cell-free system using a multi-channel dialysis device which enables the concurrent synthesis of different proteins in adequate yields under simple and reproducible conditions.
The principle of cell-free in vitro protein biosynthesis using dialysis membranes has been known for several years and essentially comprises two separate chambers (one for the reaction mixture, one for the supply solution) that are connected via a membrane with a suitable pore size. Protein synthesis takes place in the reaction chamber. The supply chamber contains a solution of all reaction components required for transcription or translation that are consumed in the reaction mixture during the process of protein biosynthesis. Due to the fact that both chambers are connected by a semipermeable membrane, consumed reaction components in the reaction chamber can be continuously replaced by new components from the supply chamber, if necessary by means of an appropriate pumping device, and consequently synthesis can be maintained over a considerably longer time period compared to a static system i.e. a corresponding reaction that takes place in a reaction vessel that is not subdivided. Corresponding methods for cell-free in vitro biosynthesis based on the continuous flow or the continuous exchange principle are described for example in U.S. Pat. No. 5,478,730, EP 0 593 757 and by Spirin et al. in Science vol. 242, 1988 p. 1162-1164.
The synthesis system used according to the document U.S. Pat. No. 5,478,730 for example contains a source of DNA or mRNA which codes for the polypeptide. In addition the cell-free synthesis system essentially contains ribosomes, tRNA, amino acids, ATP, GTP, UTP and CTP. The transcription of DNA and the translation of mRNA with the aid of tRNA results in the production of the respective polypeptide together with low molecular weight by-products and waste materials. These can pass into a supply space through a semi-permeable membrane which segregates the space containing the synthesis system from the supply space. The supply space contains a liquid which acts as a supply medium containing in particular ATP, GTP and amino acids. These components are supplied to the synthesis system through the semipermeable membrane in order to replenish materials consumed during the biosynthesis reaction. Passage through the semipermeable membrane is possible since their molecular weight is below the cut-off limit. At the same time products of the biochemical reaction and other substances with a molecular weight below the cut-off of the barrier pass from the reaction space into the supply space. According to the U.S. Pat. No. 5,478,730 the semipermeable membrane is for example an ultrafiltration membrane in the form of hollow fibre membranes.
The U.S. Pat. No. 5,478,730 contains extensive additional information on suitable compositions for the synthesis system and the supply liquid. To this extent the present invention makes reference to the prior art and in particular this U.S. patent document and the literature references cited therein. The contents thereof are incorporated into the present application by way of reference.
The European Patent 0 593 757 describes the application of coupled transcription and translation for corresponding in vitro biosynthesis methods in which essentially an RNA polymerase was additionally added to the reaction mixture.
In addition various dialysis devices and membranes are known. The main distinction is between so-called single tube and multi-channel designs. The previously described and commercially available dialysis materials that are suitable for in vitro protein biosynthesis are exclusively based on the single tube principle (e.g. EP 99 91 01 418.4; Promega Notes 1997). These dialysis aids do not enable a high throughput or the concurrent synthesis of different proteins especially without complicated purification procedures or other measures such as the denaturation of proteins that are synthesized as inclusion bodies. An additional disadvantage of dialysis devices based on the single tube principle is that they are technically difficult to operate and time consuming.
Furthermore microtitre plates containing wells which are provided with a porous membrane at the lower end are commercially available. So-called multi-channel versions are only suitable for cell culture applications or for filtration or rebuffering processes (e.g. ultra-filtration membranes from Milipore; Slide-A-Lyzer® MINI Dialysis Unit from Pierce). However, these multi-channel devices do not allow an exchange of liquids of different concentrations that are used for in vitro transcription or in vitro translation which is why they are unsuitable for in vitro protein synthesis applications with for example a continuous supply and removal of components.